Micro-organ device

ABSTRACT

A method for fabricating a micro-organ device includes printing a micro-organ on a microchip using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a scaffold, and wherein the printing is performed with the computer-aided tissue engineering system according to a predetermined pattern. A micro-organ device includes at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of application Ser. No. 12/058,227 filed on Mar. 28, 2008, which claims the benefit of U.S. Provisional Application Ser. No. 60/908,918, filed on Mar. 29, 2007.

ORIGIN OF INVENTION

The invention described herein was made in the performance of work under a NASA contract and is subject to the provisions of Section 305 of the National Aeronautics and Space Act of 1958, Public Law 85-568 (72 Stat. 435; 42 U.S.C. 2457).

BACKGROUND OF INVENTION

1. Field of the Invention

The invention relates generally to devices for testing of biologics. More particularly, this invention relates to methods and technologies for producing micro-organ devices.

2. Background Art

Testing of pharmaceuticals and biological compounds in human or animals is not always possible, at least not in the early stage. Therefore, sometimes decisions need to be made based on in vitro data. Extrapolating in vitro data (e.g., cell culture data) to the in vivo relevant conditions is often very difficult. Although pharmacokinetic principles can be used to derive some conclusions, this approach has limitations due to various reasons.

For example, cell cultures under the assay conditions may not function in the same ways as they would in the natural settings. The communication and interactions between different tissues and organs are absent. In culture, cells are typically grown at the bottom of chambers or wells. These systems may have unreaslistically high liquid-to-cell ratios. Even if the cells are grown on microcarrier beads, which more closely resemble physiological conditions, they still may not mimic physiological conditions accurately enough to provide reliable data.

For example, cell-cell interactions between different cell types may be important. The conventional cell cultures do not allow proper interactions between different cell types. U.S. Pat. No. 6,221,663 issued to Bhatia et al. discloses methods for producing co-cultures of cells in which at least two cell types are present in a micropattern configuration.

Having cell-cell interactions in a co-culture is an improvement over the traditional cell culture methods. However, in human or animals, livers often play important roles in the metabolism and bioavailability of pharmaceuticals and biological compounds. Consequently, methods of predicting human response from in vitro cell culture assays are not always reliable. Even though in vivo animal studies can provide data more relevant to human responses, animal testings are expensive, labor-intensive, and time consuming.

Therefore, there is a need for in vitro organ or system models that can mimic the in vivo organs or systems in animals. U.S. Pat. No. 5,612,188 issued to Shuler et al, discloses a multi-compartmental cell culture system. This patent is incorporated by reference in its entirety. This culture system includes components, such as culture chambers, sensors, and pumps. This system is expensive to operate and requires a large amount of space. Because this system is on such a large scale, the physiological parameters vary considerably from those found in an in vivo situation. It is impossible to accurately generate physiologically realistic conditions at such a large scale.

U.S. Patent Application Publication No. 2003/0082795 A1 by Shuler et al. discloses in vitro microscale cell culture analog devices that permit cells to be maintained in vitro under conditions similar to those found in vivo. This patent is incorporated by reference in its entirety. The microscale culture device comprises a fluidic network of channels segregated into discrete but interconnected chambers. The specific chamber geometry is designed to provide cellular interactions, liquid flow, and liquid residence parameters that correlate with those found for the corresponding cells, tissues, or organs in vivo. The fluidics is designed to accurately represent primary elements of the circulatory or lymphatic systems. In one embodiment, these components are integrated into a chip format. The design and validation of these geometries is based on a physiological-based pharmacokinetic (PBPK) model; a mathematical model that represents the body as interconnected compartments representing different tissues.

Similarly, U.S. Patent Application Publication No. 2003/0152909 A1 discloses micro-organ cultures which include isolated populations of cells having specific characteristics. These micro-organ cultures have the ability to be maintained in culture for relatively long periods of time, as well as the preservation of an organ micro-architecture which facilitates, for example, cell-cell and cell-matrix interactions analogous to those in the source organ.

These prior art organ models demonstrate the advantages of these in vitro systems for pharmaceutical or biological compounds testing. However, the prior art approaches rely on directly placing the cells in the chamber or seeding the cells and allowing them to grow in the chamber. These approaches do not always provide reproducible organs. Therefore, there remains a need for better in vitro organ devices and methods for their preparation.

SUMMARY OF INVENTION

In one aspect, embodiments of the present invention relate to methods for fabricating a micro-organ device. A method in accordance with one embodiment of the invention includes printing a micro-organ on a microchip using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a scaffold, and wherein the printing is performed with the computer-aided tissue engineering system according to a predetermined pattern.

In one aspect, embodiments of the present invention relate to micro-organ devices. A micro-organ device in accordance with one embodiment of the invention includes at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes spacing, between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

Other aspects and advantages of the invention will be apparent from the following description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic illustrating a method for fabricating a microchip for a micro-organ device in accordance with one embodiment of the invention.

FIG. 2 shows a schematic illustrating possible chemical modification reactions of silanol groups on a PDMS microchip.

FIG. 3 shows a schematic illustrating a computer-aided tissue engineering system that can be used to bioprint micro-organ devices in accordance with one embodiment of the invention.

FIGS. 4A-4C shows three micro-organ devices with different configurations of organ arrays and microfluidic channels in accordance with embodiments of the invention.

FIGS. 5A and 5B show an expanded view of a section of a micro-organ, illustrating the substructures produced with bioprinting, in accordance with one embodiment of the invention.

FIG. 6 shows a 2D cross sectional view of a 3D micro-organ in accordance with one embodiment of the invention.

FIG. 7 shows a prospective view of a 3D micro-organ device in accordance with one embodiment of the invention.

FIG. 8 shows a diagram, illustrating various microfluidic channels and interconnections on a micro-organ device in accordance with one embodiment of the invention.

FIG. 9 shows a chart of results from an activity assay using a liver micro-organ device in accordance with one embodiment of the invention.

FIGS. 10A-10E show examples of various configurations and arrays that may be produced with methods of the invention for various studies.

DETAILED DESCRIPTION

“Bioprint” or “bioprinting” as used in this description refers to a process of forming micro-organs using a computer-aided tissue engineering (CATE) system to print a micro-organ according to a predetermined design pattern. These processes will be described in more detail in the following sections.

“Microscale” as used herein refers to dimensions no greater than 10 cm, preferably no greater than 1 cm.

“Microchip” as used herein refers to a microscale support having one or more microfluidic channels and one or more micro-chambers for housing micro-organs. A microchip typically has a dimension on the order of a few centimeters.

“Microfluidic channel” as used herein refers to a channel on a microchip. Such channels typically have depth and width that are less than a few millimeters, preferably less than 1 mm.

“Micro-chamber” as used herein refers to a structure (e.g., a well or indentation) on a microchip. A micro-chamber typically has a dimension of no greater than a few cm, preferably 1 cm or less. A micro-chamber is typically for housing cells of a micro-organ.

“Micro-organ” as used herein refers to cells bioprinted on a microchip, typically in a micro-chamber on the microchip.

“Micro-organ device” or “MOD” as used herein refers to a device having at least one micro-organ on a support (such as a microchip).

Embodiments of the invention relate to methods for producing micro-organ devices (MOD) using bioprinting technology and the devices thus produced. The micro-organ devices in accordance with embodiments of the invention comprise microscale 3D tissue analogs in devices that include microfluidic channels and chambers.

In accordance with embodiments of the invention, different mammalian tissues may be mimicked on MODs for various applications, such as experimental pharmaceutical screening for efficacy and toxicity. Thus, these devices may be used to assess the beneficial and detrimental effects of a novel drug after it passes through a given metabolic pathway. For example, the therapeutic or toxic effects of a drug on any cell type in vitro may be assessed in the presence of microencapsulated liver cells to take into account the liver-dependent metabolic modifications of the candidate drug. In addition, these devices may be used to evaluate drug therapeutic benefits or toxicities. These MODs can address the need for in vitro micro-organs that can substantially replicate in vivo structure and function.

The micro-organ devices (MOD) in accordance with embodiments of the invention may be produced by direct bioprinting of specific cells, human or animal, to form micro-organs in micro-chambers of microchips. The bioprinting processes are more controllable and can produce reproducible micro-organs that also mimic the in vivo functions more closely.

The methods of the invention may include forming microchips that include microfluidic channels and micro chambers for housing micro-organs. Next, the desired organs may be bioprinted in the micro-chambers. The bioprinting may use Computer Aided Tissue Engineering (CATE) technology. CATE integrates computer-aided design (CAD) technologies and modern design and manufacturing in bioengineering and biomedicine. CAFE is used in the design, simulation and fabrication, of tissues. For more description of CATE, see e.g., http://www.mem.drexel.edu/cate.

FIG. 1 illustrates an exemplary method that can be used to fabricate a micro device of the invention. As shown in FIG. 1, a master 10 for making microchips comprises a design pattern 12 on a silicon wafer 11. The master 10 may be fabricated using techniques that are adapted from lithographic techniques commonly used in integrated circuit (IC) chip manufacturing. Examples of using the lithographic techniques to fabricate tissue/organ devices can be found in U.S. Patent Application Publication No. 2003/0082795, The design pattern 12, for example, may include fluid communication channels and tissue chambers for forming a micro-organ device. A suitable polymer reagent is then poured onto the master 10 and allowed to cure, as shown in 13, to form a replica of the master 10. Any suitable polymer reagents may be used for making the replica. One suitable example includes a class of compounds generally referred to as polydimethyl siloxanes (PDMS). Other suitable materials, for example, may include rubber, polymers, plastic, elastomers, and silicone materials (such as Formulations RTV 108, RTV 615 or RTV 118 available from General Electric, New York, N.Y.).

PDMS is a flexible elastomeric polymer that is an excellent material for microfluidic device fabrication. See, McDonald et al., “Fabrication of microfluidic systems in poly(dimethylsiloxane),” Electrophoresis 2000, 21(1), pp. 27-40. Various PDMS are commercially available, including Sylgard® 184 from Dow Corning. Sylgard is a two-part resin system containing vinyl groups (part A) and hydrosiloxane groups (part B). Mixing of the two resins under appropriate conditions (e.g., by heating at a temperature of 65° C. or higher for 2 hours) leads to crosslinking (or curing), as illustrated below:

Once the polymer cures, the flexible replica 14 can be peeled off the master, leaving the master ready to produce another replica. The replica 14 has a pattern complementary to the design pattern 12. Once the replica 14 is peeled from the mold, it is ready for assembly into a final micro-organ device. These devices may be cut to proper sizes with a standard surgical blade, and access holes may be punched, if needed, for tubings and other device inputs. For tubing inputs, a needle hole slightly smaller than that outer diameter of the tubing may be drilled so that adequate sealing can be achieved.

Another useful property of PDMS is that its surface can be chemically modified in order to obtain the interfacial properties of interest. See, Makamba et al., “Surface modification of poly(dimethylsiloxane) microchannels,” Electrophoresis 2003, 24(21), 3607-19. One suitable method to covalently functionalize PDMS is to expose it to an oxygen plasma, whereby the surface of Si—CH₃ groups along the PDMS backbone are transformed into Si—OH groups by the reactive oxygen species in the plasma. For example, the PDMS surface may be exposed to O₂ plasma (100 mW radiofrequency, O₂) for 30 seconds to convert the surface methyl silane groups into silanol groups.

These silanol surfaces can then be easily transformed with other alkoxysiloxanes to yield any different chemistries, some of which are illustrated in FIG. 2, to facilitate further modification or reaction on the replica. The modified PDMS surface, which now has modified siloxane groups, can be reacted and bonded with another object 15 that contains silanol groups, such as a glass surface, another PDMS, or the like. Alternatively, the original PDMS replica from the polymerization reaction shown above (which contains methyl siloxane groups) can be reacted directly with silanol groups from the other object 15. With either approach, a microchip having microfluidic channels and micro-chambers can be readily prepared.

In accordance with embodiments of the invention, the organs to be placed in the micro-chambers of MODs may be fabricated or printed using a computer-aided tissue engineering system (CATE). FIG. 3 shows a computer-aided tissue engineering (CATE) system 30 that includes a computer 31 for controlling one or more syringes 32. The computer 31 includes one or more CPU and memories, which store a CATE program. A CATE program typically includes computer-aided design/engineering/manufacturing (CAD/CAE/CAM) software The CATE system also includes a precision deposition system (which includes the syringes 32 shown in FIG. 3) for biomanufacturing of tissues or organs. Cells for bioprinting are suspended in a solution containing extracellular matrix (e.g., collagens) and/or scaffold materials (e.g. alginates) and loaded in the syringes 32 for bioprinting the micro-organs 35.

Using the methods and systems illustrated in FIGS. 1 and 3 (or variation thereof), microchips (for micro-organ devices, MOD) in accordance with embodiments of the invention can be readily prepared. After loading the cells in the micro-chambers (using the bioprinting process that will be described later), micro-organ devices (MOD) of the invention are obtained. These MODs may have various configurations of micro-chambers (housing micro-organs) interconnected by microfluidic channels to simulate the fluid communications between these organs in vivo, FIGS. 4A-4C show some schematics that illustrate some micro-organ devices in accordance with embodiments of the invention.

FIG. 4A shows a schematic of a micro-organ device 40 that includes a single micro-organ 41 in communication with two micro-channels 42. This device is useful for assessing the effects (beneficial or toxic) on a particular organ and/or the mechanisms underlying these effects. For example, the micro-organ 41 may be a liver or kidney, and the device may be used to assess various drugs toxicities to the liver or kidney.

In addition, this device 40 may be used to study metabolism of a drug by a particular organ, such as liver. Livers play critical functions in various biological pathways involving xenobiotics, such as drugs. The functions of livers may include converting a pro-drug into an active drug, converting a drug into a metabolite that may be more active or less active, converting a toxic compound into a soluble form for excretion (detoxification), etc.

Therefore, a micro-organ device having a micro-liver will allow one to use such a device to study these reactions without resorting to animal or human tests. In such assays, a drug or test compound may be supplied in a solution from one end of a micro-channel 42. The capillary effect and gravity may be used to draw the solution into the micro-chamber, where the micro-liver 41 is located, and out the other end of the micro-channel 42. If necessary, pumps, pressure or vacuum may also be used to circulate the fluids in the microfluidic channels. The metabolites or modified drugs collected from the outlet may be analyzed to elucidate the liver metabolism pathways or to study the enzyme activities. In addition, the modified drugs may be used to test on a target micro-organ. Or the like.

FIG. 4B shows a micro-organ device 47 having two micro-organs 41 and 43. The micro-organ device 47 also includes several micro-channels 42 for flowing solutions containing, compounds to be tested. This particular micro-organ device 47 is suitable for studying the interactions between the micro-organs 41 and 43 or the impacts of the presence of one micro-organ (e.g., 41) on the other (e.g. 43) when a drug is administered.

For example, the micro-organ 41 may be a micro-liver, while the micro-organ 43 may be the intended target for the drug (e.g., bone). When a drug A is passed through the micro-organ 41 (e.g., liver), it may be converted into a metabolite A′, which may be more active or less active than the original drug. The metabolite A′ then acts on the target organ (e.g., the micro-organ 43). After acting on the target organ, the metabolite A′ may be further metabolized to a new metabolite A″. With such a device, the pharmacokinetic and pharmacodynamic studies will be performed under conditions more closely related to the in vivo conditions than using a cell culture or a single organ device.

A micro-organ device of the invention may include any number of micro-organs. With a proper design of micro-chambers and interconnecting micro-channels, the various interactions between the organs in human may be replicated. FIG. 4C illustrates another embodiment of the invention, which shows a micro-organ device 49 that includes three micro-organs 41, 43, and 45. The three micro-organs 41, 43, and 45 are interconnected via micro-channels 42. In addition, two curvy arrows signify that crosstalk exists between micro-organs 41 and 43. The communication between the micro-organ 41 and 43 may be via one or more microfluidic channels. The crosstalk may be mediated by the modified compound (i.e., metabolite) after conversion by a micro-organ. Alternatively, the crosstalk may be mediated by paracrine factors, hormones, or other factors that are secreted by the cells of the micro-organs in response to a stimulus, e.g., the drag or a metabolite of the drug.

Note that the examples shown in FIGS. 4A-4C are for illustration only. One of ordinary skill in the art would appreciate that embodiments of the invention are not so limited and that modifications or variations from those shown are possible without departing from the scope of the invention. For example, the micro-channels in these illustrations are shown to have open ends. Some of these openings may be interconnected to form loops or circuits. Similarly, the multiple micro-organs shown in FIG. 4C are arranged and connected by microfluidic channels) in series (i.e., aligned sequentially). Embodiments of the invention may also include multiple micro-organs that are connected by microfluidic channels in “parallel” or in a web.

The microfluidic channels illustrated in the devices of FIGS. 4A-4C may be on the order of tens to hundreds of micrometers (μm) wide, while the micro-chambers that house the micro-organs may have a dimension as large as 1 cm or more. If the micro-organ comprises a solid mass of millimeter (mm) or centimeter (cm) in size, the cells in the interior of the micro-organ will not be able to exchange efficiently with the media in the microfluidic channels. In general, to ensure effective diffusion exchange, cells are preferably not more than 500 μm away from the microfluidic channels, in order to ensure efficient diffusion exchanges, a micro-organ in accordance with embodiments of the invention may comprise substructures, at least one dimension of which is preferably no larger than 1 millimeter (mm), more preferably less than 500 μm. Such micro-organs may be prepared according to the processes shown in FIGS. 5-8, using the CTAE system shown in FIG. 3.

FIG. 5A shows an expanded view of a small section of a micro-organ of the invention. In FIG. 5A, two substructures 51 and 52 of a micro-organ 50 are shown to have been fabricated (bioprinted) in an interdigitated configuration, leaving spaces between them. The open spaces between the two sub-structures 51 and 52 are designed to form the microfluidic channels 53. FIG. 5B shows a side view of the substructures of FIG. 5A, illustrating the 3D structure comprising multiple layers. Although only three layers of the substructures are shown in this illustration, a micro-organ of the invention may comprise many more layers, which will be discussed with reference to FIGS. 6-8. In this particular example, each Layer is about 200 μm thick. Such micro scale is desirable because it ensues that cells at the interior of the micro-organ can efficiently exchange with the media (by diffusion) in the micro-channels.

The interdigitated substructures and the multi-layer stacking may be printed using the CATE system shown in FIG. 3. In bioprinting a micro-organ, cells for the micro-organ are suspended in a solution containing one or more extra-cellular matrix or scaffold materials. The solution is put in a syringe controlled by a computer for printing the micro-organ according to a design. The printing process may be performed one layer at a time until the desired number of layers is achieved.

The interdigitated configuration of the substructures 51 and 52 and the space between them that forms the microfluidic channels 53 are more clearly shown in a top view illustrated in FIG. 6. This top view shows a section of an entire micro-organ 60 comprising many repeated layers (interdigitated folds) of substructures 51, 52, and the microfluidic channel 53 to achieve a desired dimension (e.g., 1 cm).

The 2D top view shown in FIG. 6 represents a layer (section) of a 3D micro-organ. FIG. 7 shows a perspective view of a 3D micro-organ 71 that comprises many of the 21) sections shown in FIG. 6. FIG. 7 shows that the 3D micro-organ 71 is disposed in a micro-chamber of a micro-organ device (MOD) 70. The micro-organ device (MOD) 70 also includes a microfluidic channel 72 with an inlet 72 a and an outlet 72 b. As noted above, the MOD 70 may be made of any suitable materials, such as PDMS. The MOD 70 is shown to be supported on a chip 75, which may be a silicon wafer, glass or plastic and have a dimension on the order of one to several centimeters.

As shown in FIGS. 4B and 4C, a micro-organ device (MOD) of the invention may include more than one micro-organ, which may be interconnected by one or more microfluidic channels. FIG. 8 shows a diagram, illustrating an MOD 80 in accordance with one embodiment of the invention that comprises two micro-organs 81, 82 connected by a network of microfluidic channels 83. The network of microfluidic channels 83 is shown to include various valves (V), reservoirs (R), pumps (P), and detector (D).

In the example shown in FIG. 8, the chip footprint may be in the range of 1-5 cm×1-5 cm. The fluid channels may be about 25-50 μm deep and about 100-350 μm wide. The fluid channels in the electrophoresis section may be smaller, e.g., about 50 μm wide. The valves (V) may be controlled by pneumatic control lines, which serve to actuate the valves for controlling fluid flow and fluid pumping. The pneumatic control lines may be 10 μm deep and 200 nm wide, for example. Note that these dimensions are for illustration of this particular example. One of ordinary skill in the art would appreciate that MOD in accordance with embodiments of the invention may also be prepared in other sizes and dimensions.

The pumps shown in FIG. 8 may be peristaltic pumps or syringe pumps. The detector may be any suitable detector (optical, UV, fluorescence, IR, laser, etc.) for detecting or assessing drug conversion or metabolite analysis. The MOD shown in FIG. 8 also includes an electrophoresis separation and detection section, which may be used to analyze any metabolites or converted drugs.

The methods and technologies for bioprinting of MODs in accordance with embodiments of the invention do not adversely impact the viability of the cells in one example, using mouse hepatocytes (ATCC, CRL-2254) encapsulated in alginate hydrogels, it was found that the resultant micro-organ retained 79% viable cells. A liver micro-organ thus prepared was found to produce a higher amount of urea than the same number of hepatocytes cultured on tissue culture plastic, suggesting that the cells in the MODs of the invention are healthier.

More importantly, micro-organ devices prepared with these methods are found to be able to perform functions similar to the in vivo functions of the corresponding organ. For example, FIG. 9 shows a chart illustrating results form the conversion of 7-ethoxy-4-trifluoromethylcoumarin (HFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) by a liver micro-organ device in accordance with one embodiment of the invention. EFC is converted to HFC by liver cytochromes P450 (P450s). Liver cytochrome P450 enzymes are involved in drug metabolism. EFC has weak or no fluorescence, while HFC has strong fluorescence. Therefore, this system provides a convenient assay for the drug metabolizing enzymes in the liver. Liver MODs in accordance with embodiments of the invention are found to convert EFC to HFC at rates ranging from 1 to 10 picomoles/hr/10⁴ cells. Results shown in FIG. 9 suggest the MODs of the invention can have functions like those of the in vivo organs. Therefore, these MODs can be used to study drug metabolism or other applications.

Methods and technologies in accordance with embodiments of the invention can provide direct and reproducible biopatterning or bioprinting of human or animal cells, extracellular matrix and scaffold materials directly on microfluidic microchips in in vivo-like architectures and arrays. With these methods, it becomes possible to design and test various drugs in a more in vivo-like platform that includes proper inter-communications between different organs. FIGS. 10A-10E show a few diagrams illustrating some configurations and applications that are possible with MODs of the invention. The embodiment shown in FIG. 10A includes a single micro-organ. This embodiment will be useful for studying the effects of drugs on the target organ, biological functions of the target organ, or to use the target organ to convert the drugs to new metabolites, as illustrated in FIG. 9.

FIG. 10B Shows an MOD with two target organs, the first target organ (e.g., liver) converts a drug (D_(x)) into a new compound (D_(x)′), which then acts on the second target organ. This embodiment can be used to study the conversion of a drug to a new metabolite and its effect on a target organ.

FIG. 10C shows an MOD having two target organs. The two organs interact with each other. Thus, a drug (D_(x)) converted by the first target organ to a new compound (D_(x)′) acts on the second target organ, while another drug/compound (D_(Y)) converted by the second target organ to a new metabolite (D_(Y)′) can have an effect on the first target organ.

FIG. 10D shows an MOD having two organs that can independently regulate a third organ. These two organs may convert drugs (D_(A). D_(B)) to metabolites (D_(A)′, D_(B)′) that affects a third organ. Alternatively, these two organs may secrete biological effectors (D_(A)′, D_(B)′) in response to the stimuli of drugs (D_(A), D_(B)) that act on a third organ.

FIG. 10E shows an MOD having two organs that may jointly regulate a third organ. In this case, the metabolites or biological effectors (D_(A)′, D_(B)′) produced by the first two organs may need to be combined (e.g., dimmer formation, etc.) in circulation before they can act on the third organ. Alternatively, the two metabolites or biological effectors (D_(A)′, D_(B)′) may act on an intermediate organ, which then produces a signal to regulate the third target organ.

The various embodiments shown in FIGS. 10A-10E are for illustration only. One of ordinary skill in the art would appreciate that various other configurations may be made with MODs of the invention to suit the purposes of the studies.

Advantages of embodiments of the invention may include one or more of the followings. Methods in accordance with embodiments of the invention use direct biopatterning and bioprinting on microfluidic microchips. These methods eliminate or minimize variations in local seeding densities and also minimize selection pressures that favor more aggressive cells. Direct bioprinting also enables precise and simultaneous coupling of multiple microfluidic channels on one microchip with those on another.

These methods also allow better control of volumetric and quantitative accuracy for specific applications (such as pharmacodynamics, pharmacokinetics, and toxicity studies). They also enable reproduction of in vivo circulation profiles within and between micro-organs.

The MODs in accordance with embodiments of the invention include in vivo-like micro-organs, and may include unique micro-environments and fluid pathways that mimic the in vivo situations. Methods of the invention provide the reproducibility and quality control that are lacking in the current art; and use simultaneous biopatterning or bioprinting of the cells, extracellular matrix and scaffold directly on the microchip in in vivo-like architect.

The MODs require little resources for investigation of in vivo toxicology and pharmacological effects with high extrapolation to humans. These MODs may be used both in space and on Earth as a drug screening system with human cell micro-organs to supplement/reduce animals studies, and to increase success of clinical trials.

The MODs of the invention may be used in drug screening to supplement/reduce animal studies, and to increase success of clinical trials with new drugs.

The micro-organ-devices (MOD) are designed to mimic the complex in vivo microenvironment by replicating the in viva micro-organ structure, extracellular matrix composition and architecture, and the in viva fluid flow. One or more micro-organs are created by suspending human or animal mammalian cells in a specific soluble extra cellular matrix (ECM) and scaffold, and printed as three-dimensional micro-organs. The micro-organs are directly printed and arrayed as single or multi-organs on micro-fabricated microfluidic devices. The microfluidic device houses the micro-organs, controls and directs the perfusion of cell culture medium and specific soluble factors such as investigative drugs, and chemicals. Fluids are circulated through the micro-organs to maintain viability and to deliver specific soluble factors and molecules. Biopatterning cells, ECM and scaffold directly onto the microchips provide excellent reproducibility and quality control.

The Micro-Organ-Devices (MOD) in accordance with embodiments of the invention address the need for an automated, high-throughput in vitro system that (i) reproduces selected in vivo micro-organs and their fluid pathways, (ii) uses a simultaneous patterning or bioprinting of cells, extracellular matrix and scaffold materials directly on the microchip in in vivo-like architectures and arrays, (iii) provides the required reproducibility and quality control for high throughput and long term operation, and (iv) requires minimal resources (power, mass, fluids).

The MODs in accordance with embodiments of the invention provide in vitro assay systems using human cell-based biopatterned micro-organs with a micro-fabricted, microfluidic chip that more precisely simulates in vivo (especially human) responses to pharmaceuticals. Such an alternative assay platform would enable a greater proportion of candidate drugs to “fail early” prior to clinical trials than the current approach using animal tests, potentially reducing costs and maximize resources and success by accurately selecting a smaller number of candidates for in vitro and in vivo drug metabolism and drug target validation.

The MODs in accordance with embodiments of the invention can be automated, require minimal footprints and power requirements, use micro-volumes of fluids and generate minimal amounts of wastes, and can provide high throughput and parallel analyses on the same chip.

While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims. 

What is claimed is:
 1. A micro-organ device, comprising: at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.
 2. The micro-organ device of claim 1, wherein the cells are liver cells.
 3. The micro-organ device of claim 1, wherein at least one micro-chamber each housing a micro-organ comprises two micro-chambers housing two different micro-organs.
 4. The micro-organ device of claim 3, wherein one of the two different micro-organs comprises liver cells.
 5. The micro-organ device of claim 1, wherein the configuration of the micro-organ comprises a pair of interdigitated substructures. 